Reversible protein phosphorylation is a major mechanism for the co-ordinated control of many fundamental cellular functions in eukaryotic organisms, including metabolism, growth, and differentiation. The phosphorylation status, and consequently the activity, of specific target proteins is regulated by the opposing actions of protein kinases and protein phosphatases. Generally, these enzymes are specific either for serine/threonine or for tyrosine phosphoacceptors, although some dual specificity kinases and phosphatases have also been described. The importance of phosphorylation cascades is reflected by the finding that many kinases, phosphatases, and the signal transduction pathways in which they participate have been highly conserved during the course of evolution.
It has become increasingly clear over the last 10 years that the products of most of the genes involved in cellular transformation and cancer i.e. oncogenes and tumour suppressor genes are components of signal transduction pathways (Hanahan and Weinberg, 2000, Cell, 100, 57-70). Protein kinase B (PKB) has been well established as an important signalling intermediate, and its de-regulation has been implicated in the development of human cancer and diabetes (reviewed in Brazil and Hemmings, (2001), Trends Biochem Sci 26: 657-64).
Protein kinase B (c-Akt/PKB) is an ubiquitous Ser/Thr protein kinase which has a complex mechanism of regulation yet to be completely resolved (Downward et al., 1998, Curr. Opin. Cell Biol., 10, 262-267; Coffer et al., 1998, Biochem. J., 335, 1-13; Kandel and Hay, 1999, Exp. Cell Res., 253, 210-229; Vanhaesebroeck and Alessi, 2000, Biochem. J. 346 Pt 3, 561-576). Upon cell stimulation, localized generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the plasma membrane recruits PKB to the membrane through its N-terminal pleckstrin homology (PH) domain. PKB is then activated by phosphorylation on two regulatory sites: Thr308 in the activation loop in the kinase domain and Ser473 in the hydrophobic C-terminal regulatory domain (Alessi et al., 1996, EMBO J., 15, 6541-6551).
In cells lacking the tumour suppressor PTEN (a lipid phosphatase), PKB is more active (Cantley and Neel, 1999, Proc. Natl. Acad. Sci. USA, 96, 4240-4245; Vazquez and Sellers, 2000, Biochim. Biophys. Acta, 1470, M21-M35), as a result of the increase in phosphorylation at Thr 308 and Ser 473. The kinase PDK1, a kinase that contains a PH domain, has been shown to be able to phosphorylate PKB at Thr-308 in vivo (Alessi et al., 1997, Curr. Biol., 7, 261-269; Stokoe et al., 1997, Science, 277, 567-570; Stephens et al., 1998, Science, 279, 710-714). Despite PDK1 being identified as the PKB Thr308 kinase, the kinase responsible for phosphorylating Ser473 in vivo, often referred to as PDK2 or Ser473 kinase, remains elusive.
Several kinases have been reported to possess Ser473 phosphorylating activity, including mitogen-activated protein kinase-activated kinase-2 (MAPKAPK-2) (Alessi et al., 1996, EMBO J., 15, 6541-6551), integrin-linked kinase (Delcommenne et al., (1998), Proc Natl Acad Sci USA 95: 11211-11216), PDK1 (Balendran et al., (1999), Curr Biol 9: 393-404; WO 00/36135) and PKB (Toker and Newton, (2000), J Biol Chem 275: 8271-8274). However, evidence has been presented which argues against these kinases as the physiological PKB Ser473 kinase. For example, activation of MAPKAPK-2 is PI3-kinase-independent, whereas PKB Ser473 phosphorylation is sensitive to PI 3-kinase inhibitors (Alessi et al., 1996, EMBO J., 15, 6541-6551). PDK1-null cells undergo Ser473 phosphorylation, suggesting that PDK1 is not required for Ser473 phosphorylation (Williams et al., (2000), Curr Biol 10: 439-448). Furthermore, insulin-stimulated PKB Ser473 phosphorylation does not require activation of PDK1 or PKB, as Ser473 phosphorylation is not sensitive to staurosporine treatment, which inhibits PDK1 and therefore PKB activity (Hill et al., (2001), J Biol Chem 276: 25643-25646).
Another kinase, ILK, was shown to phosphorylate glycogen synthase kinase-3, as well as Ser473 of PKB (Delcommenne et al., (1998), Proc Natl Acad Sci USA 95: 11211-11216). However, it has also been suggested that ILK influences PKB phosphorylation indirectly, as overexpression of certain kinase domain mutants can mimic wild type ILK in inducing Ser473 phosphorylation (Lynch et al., (1999), Oncogene 18: 8024-8032). Moreover, a physiological role of ILK in regulating PKB phosphorylation has been questioned since ILK knockout in Drosophila melangaster shows a phenotype more similar to the integrin knockout than to the PKB knockout (Zervas et al., (2001), J Cell Biol 152: 1007-1018).
Developing methods to regulate PKB Ser473 kinase activity requires sources of purified PKB Ser473 kinase. Purified PKB Ser473 kinase would, for example, be useful in developing and testing assays for measuring PKB Ser473 kinase activity, to evaluate the assay and for use as a standard in the assay. Assays for PKB Ser473 kinase are useful in screening for modulators of PKB signaling or other signaling pathways dependent on PKB Ser 473 kinase activity. Purified PKB Ser473 kinase would be more useful than crude cells to identify and test modulators, inhibitors or activators of PKB Ser473 kinase activity in in vitro assays. Such modulators of PKB Ser473 kinase activity would be useful in the treatment of a condition associated with PKB signaling pathways, for example, an anomaly in cell growth or with an anomaly in insulin regulation, such as cancer, diabetes or other PKB-dependent conditions, such as neurodegenerative conditions or erectile dysfunction.
Moreover, purified PKB Ser473 kinase would facilitate a thorough biochemical analysis of the kinase's mechanism, which may provide insight for development of mechanism-based regulators. Purified PKB Ser473 kinase also would be useful in the preparation of antibodies against PKB Ser473 kinase, including phospho-specific antibodies. Such antibodies would in turn be especially useful as reagents to purify human PKB Ser473 kinase and may be useful in cancer diagnosis or prognosis. Purified PKB Ser473 kinase also will help provide amino acid sequence information useful in designing various mutants or fragments of the PKB Ser 473 kinase.
While there is a need for characterization and purification of PKB Ser473 kinase, the purification of the human enzyme has posed technical challenges. Human cells possess high levels of other kinases that might have chromatographic purification properties similar to the PKB Ser473 kinase making purification of PKB Ser473 kinase from human cells particularly difficult. In addition, a specific assay had to be developed to follow the PKB Ser473 kinase activity responsible for PKB phosphorylation in vivo. Thus, there is a need for purified PKB Ser473 kinase and purified human PKB Ser473 kinase, in particular.